Method of treating or inhibiting the development of brain inflammation and sepsis

ABSTRACT

A leukotriene C4 and D4 antagonist is used to treat or inhibit brain inflammation and sepsis by acting to inhibit increased capillary permeability and white blood cell extravasation. Potential candidate compounds can be screened in a non-human mammal before or after administration of an inflammation inducing agent into the subarachnoid space by determining their ability to inhibit increased capillary permeability and white blood cell extravasation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.10/721,742, filed Nov. 26, 2003, which claims the benefit of priorityunder 35 U.S.C. §119(e) from U.S. provisional application Nos.60/429,558, filed Nov. 29, 2002, and 60/496,677, filed Aug. 21, 2003,and claims the benefit of priority under 35 U.S.C. §119 (a-d) toJapanese patent application no. 142759, filed May 20, 2003, the entirecontents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for treating or inhibiting thedevelopment of a disease, disorder or condition, which is associatedwith increased capillary permeability and white blood cellextravasation, such as brain inflammation and sepsis.

2. Description of the Related Art

Arachidonic acid (AA) is released from phospholipid in the cell membraneto the cytoplasm in response to a number of insults such as mechanical,thermal, chemical, bacterial and other insults, and its products (theeicosanoid compounds prostaglandins and leukotrienes) have been found tobe biologically important in a number of ways. Most of the eiconosaidcompounds tend to aggravate inflammatory, pain, and fever responses, andthey have been the targets of extensive research on anti-inflammatoryand analgesic drugs. For example, anti-inflammatory steroids such ascortisone function by suppressing the phospholipase enzymes thatgenerate arachidonic acid from membrane phospholipids. Pain-killers suchas aspirin and ibuprofen act by blocking to some extent thecyclooxygenase enzymes that control the conversion of arachidonic acidto the eicosanoids, prostaglandins, prostacyclins, and thromboxanes.

Additionally, it is known that prostaglandins and leukotrienescontribute to the genesis of inflammation in both the peripheral andcentral nervous system (CNS). Despite studies done over three decades,exactly how prostaglandins contribute to inflammation remains unclear.In contrast, recent studies using leukotriene receptor antagonistsindicate that leukotrienes might play a major role in this process.

Leukotrienes are potent lipid mediators and are divided into twoclasses, based on the presence or absence of a cysteinyl group.Leukotriene B₄ does not contain such a group, whereas leukotriene C₄,D₄, E₄ and F₄ are cysteinyl leukotrienes. These compounds have beenrecognized as inflammatory agents since the early 1980's (von Sprecheret al., 1993 and Piper, 1984).

In the 1990's, various drugs known as “leukotriene antagonists”, whichcan suppress and inhibit the activity of leukotrienes in the body, wereidentified. The term “leukotriene antagonist” is used herein in theconventional medical sense, to refer to a drug that suppresses, blocks,or otherwise reduces or opposes the concentration, activity, or effectof one or more subtypes of naturally occurring leukotrienes. However,such leukotriene antagonists can be classified into two different groupsbased on a difference in mechanism of action, one which to suppresses5-lipoxygenase and the other which competitively antagonizes thereceptor for leukotriene. Pranlukast, which is one leukotrieneantagonist, acts strictly at the leukotriene C4 and D4 receptor level.

Although some leukotriene receptor antagonists have been disclosed foruse in treating brain inflammation (e.g., J63258-879-A, JO2-169583-A,WO9959964-A1, EP-287-471-A), all of the disclosed leukotriene receptorantagonists are presumed to pass the blood-brain barrier (BBB) becauseit is conventional wisdom that a molecule must be able to pass theblood-brain barrier in order to reduce or inhibit brain inflammation, orto treat disorders of the brain resulting from brain inflammation(Wilkinson et al., 2001). As leukotriene C₄ and D₄ receptor antagonistsdo not pass the blood-brain barrier, insofar as is known, theleukotriene C₄ and D₄ receptor antagonists have never previously beenused to treat or prevent brain inflammation except for a neuroprotectiveeffect of pranlukast (ONO-1078), a leukotriene receptor antagonist, onfocal cerebral ischemia in rats (Zhang et al., 2002) and in mice (Zenget al., 2001). The leukotriene C₄ and D₄ receptor antagonists are,however, commonly used to treat asthma. The leukotriene C₄ and D₄receptor antagonist pranlukast is used clinically as an anti-asthmaticdrug and is known to have few side effects. Pranlukast does not pass, orpasses the blood-brain barrier at most at a very minimal level, akin tothe other antagonists such as zafirlukast and montelukast. Otheroff-label uses have been suggested for these compounds, includingtreatment of allergic diseases (Shih, U.S. Pat. No. 6,221,880) and foruse in treating migraine and cluster headaches (Sheftell et al., U.S.Pat. No. 6,194,432).

Generally, capillaries are lined with endothelial cells that havevarious openings, such as intracellular clefts, fenestrae andpinocytotic vesicles. Unlike these general capillaries, braincapillaries are characterized by the relative absence of these openingsbetween endothelial cells, but instead have tight junctions originatedfrom the periphery. Furthermore, central capillaries are surrounded byastroglia cells, which are disposed over these tight junctions ofendothelial cells (originating from the periphery). The blood-brainbarrier (BBB) is a capillary barrier comprising a continuous layer oftightly bound endothelial cells. These cells permit a low degree oftransendothelial transport, and exclude molecules in the blood fromentering the brain on the basis of molecular weight and lipidsolubility, as described in Neuwelt (1980). For example, the blood-brainbarrier normally excludes molecules with a molecular weight greater than180 daltons. In addition, the lipid solubility of molecules is a majorcontrolling factor in passage through the blood-brain barrier.

The function of the blood-brain barrier is to maintain the homeostasisof the neuronal environment. Small molecules (M.W.<200 daltons) having ahigh degree of lipid solubility and low ionization at physiological pHfreely pass through the blood-brain barrier. In addition, theblood-brain barrier allows water to move in either direction in order tomaintain equal osmotic concentrations of solutes in the extracellularcerebral fluid.

The unique biological aspect of the blood-brain barrier is an importantfocus in treating central nervous system (CNS) disorders. While theinterendothelial junctions between the cells of the blood-brain barrierare normally designed to keep potentially noxious substances away fromthe brain, this condition changes during inflammation. In other words,the permeability of the blood brain barrier increases. Braininflammation, e.g., due to stroke or physical head injury, is a seriousmedical problem causing much human misery.

The therapeutic challenges posed by brain inflammation have been tackledusing the following approaches: (1) Osmotherapy, i.e., reducing theintracranial pressure by osmotic withdrawal of water from the braintissue by intravenously administering such substances as mannitol,glycerol, urea to increase the osmolality of the blood brain barrier.Disadvantages include side effects such as electrolyte disturbances andrenal failure. (2) Steroid therapy that reduces the local capillaryleakage and global metabolic depression by means of compounds such asdexamethasone which easily crosses the blood brain barrier because ofits lipid solubility. Disadvantages are manifold, such asgastrointestinal bleeding, electrolyte disturbances, hyperglycemia,reduction of immunocompetence, increased metabolic needs, and mentaldisturbances. (3) Nonsteroidal anti-inflammatory drugs that reduce localcapillary leakage, such as indomethacin, probenecid and ibuprofen whichcross the blood brain barrier. A disadvantage is that thepharmacological effect is not certain. (4) Anti-hypertension drugs thatreduce the capillary leakage by lowering filtration pressure, by meansof, e.g., nitroprusside. Disadvantages are the reduction of cerebralperfusion pressure and the changes for the worse for brain inflammationdue to the increased capillary permeability of the blood brain barrier.However, none of these treatments improves brain inflammation.

To add to the difficulties faced by the clinician in treating braininflammation, this condition does not present a unitary symptomatology.The inflammatory response in the brain occurs in three distinct phases,each apparently mediated by different mechanisms. First, there is anacute transient phase characterized by local vasodilation and increasedcapillary permeability. This is followed by a delayed, subacute phase,most prominently characterized by infiltration of leukocytes andphagocytic cells. Finally, a chronic proliferative phase sets in, inwhich necrosis of brain cells occurs, and glia cells appear wheresubsequently, however, its original function is lost.

Brain inflammation can be assessed by various techniques such ashistochemistry and electron microscopy. However, the most significantparameter to quantify is probably the development of brain edema.Therefore, another approach has been to measure the amount of edemadeveloped in injured tissue. Edema results from the influx of watercaused by inflammation and is observed clinically as swelling. This canbe quantified by comparing tissue before and after desiccation. The dryweight remaining after drying enables calculation of the amount of fluidevaporated. The fluid evaporated is the amount of edema that was formed.

Another common laboratory technique is to determine the influx of awater-soluble dye such as Evans blue albumin into the central nervoussystem. By coupling this to a fluorescent technique, the distribution ofedema can be measured. More sophisticated and expensive methods such asPET (Positron Emission Topography), CT, MRI (Magnetic Resonance Imaging)as well as radioscintigraphy have been used as well.

However, the above methods can assess only the edema that is formed bythe increased permeability of the blood-brain barrier, but they cannotassess the infiltration of leukocytes and phagocytic cells into thecentral nervous system. Overall, edema plays a very serious role in thepathology of brain inflammation by increasing the intracranial pressure,leading to damage of the brain tissue. Additionally, the increasedpermeability of blood vessels brings about brain edema, and then,infiltration of white blood cells (WBC) is induced, resulting in a moreserious pathological condition, since lysosomal enzymes such ascollagenase and esterase damage brain tissue directly. Therefore,determination of the changes in permeability of the blood brain barrierby means of measuring the cerebrospinal fluid (CSF) volume and the WBCcount in CSF is essential.

As mentioned above, it has long been believed that in order to beeffective in the brain, a drug must be able to cross the blood-brainbarrier. Conventional targeting strategies have sought to circumventthis barrier either directly or indirectly, by administering prodrugswhose metabolites do cross the barrier or by attempting to disrupt theintegrity of the blood-brain barrier in some way. See, e.g., Pardridge,(2002). To date, however, such a clinically effective agent has neverbeen reported (Wilkinson et al., 2001).

There is therefore a need for a treatment that can be used on both anacute and a semi-acute basis to treat and inhibit the development ofbrain inflammation. The ideal compound or compounds would have minimalside effects including minimal invasiveness into the brain tissue.Additionally a method for monitoring the changes in WBC infiltrationinto the cerebrospinal fluid in animal models of brain inflammationwould also be desirable.

Despite advances in supportive care and medical technology, themortality rate from sepsis remains high. Sepsis is the most common causeof death in non-cardiac intensive care units, and its incidence appearsto be rising. Over the last two decades, the prevailing belief has beenthat much of the morbidity and mortality of sepsis is attributable tothe host's extreme inflammatory response to bacteria or bacterialproducts. Indeed, sepsis is defined clinically as the presence of two ormore conditions from the group making up what is known as the “SystemicInflammatory Response Syndrome” (SIRS), manifested in response to avariety of severe clinical insults, these conditions being: a bodytemperature higher than 38° C. or lower than 36° C.; a heart rategreater than 90 beats per minute (bpm); a respiratory rate greater than20 breaths/min. or PaCO₂ less than 32 torr (4.3 kPa); a white blood cellcount of greater than 12,000 cells/mm³ (leukocytosis), or less than4,000 cells/mm³ (leukopenia), or 10% of the total cell count beingimmature neutrophils or band neutrophils. This information is summarizedin Table 1. TABLE 1 Infection: Microbial phenomenon characterized by aninflammatory response to the presence of microorganisms or the invasionof normally sterile host tissue by those organisms. Bacteremia: Thepresence of viable bacteria in the blood. Sepsis: The systemic responseto infection or trauma. This systemic response is manifested by two ormore of (SIRS) conditions as a result of infection. Severe sepsis:Sepsis associated with organ dysfunction, hypoperfusion or hypotension.Hypoperfusion and perfusion abnormalities may include, but are notlimited to lactic acidosis, oliguria or acute alteration of mentalstatus. Septic Shock: Sepsis with hypotension, despite adequate fluidresuscitation, along with the presence of perfusion abnormalities thatmay include, but are not limited to lactic acidosis, oliguria or acutealteration of mental status. Patients who are on inotropic orvasopressive agents may not be hypotensive at the time that perfusionabnormalities are measured. Sepsis induced hypotension: A systolic BP of<90 mm Hg or of >40 mm Hg from baseline in the absence of other causesfor hypotension. Multiple Organ Dysfunction Syndrome: Presence ofaltered organ function in an acutely ill patient such that homeostasiscannot be maintained without invention.

In pre-clinical animal studies, agents designed to limit thisinflammatory response observed in sepsis have shown some initialpromising effects. However, this initial promise has not been borne outin subsequent clinical investigations. Two main approaches have beentaken: (1) Use of anti-inflammatory therapies; and (2) Use ofanti-endotoxin therapies.

With respect to the use of anti-inflammatory therapies, at least threedifferent types of agents have been found to directly limit theproduction or biologic effects of pro-inflammatory mediators. The agentsof interest are: (1) steroids such as glucocorticoids; (2) antagonistsor blockers of such pro-inflammatory cytokines as TNF-α andInterleukin-1β; and (3) antagonists or blockers of products generatedduring inflammation, such as bradykinin, or inflammatory mediators suchas prostaglandin and platelet-activating factor (PAF).

With respect to steroids, such as glucocorticoids, a large dose ofhydrocortisone has been shown to exacerbate an inflammatory response orto have no effect, but a smaller dose appears to have a beneficialeffect even though it is not a sufficiently effective remedy for sepsis.

With regard to the blocking of specific cytokines, such as TNF-α andInterleukin-1β, it has been observed that despite promising results fromanimal studies, monoclonal antibodies to TNF-α or soluble TNF-αreceptors have been shown to have no effect in clinical trials. Further,interleukin-1βreceptor antagonists appear to have no beneficial effectin clinical trials.

Finally, it also appears that other mediator specific-inflammatorytherapies including platelet-activating factor (PAF), bradykinin andprostaglandin, and the use of antagonists thereto were found to have noeffect in clinical trials.

The second strategy targets bacterial products in the circulation withthe expectation that neutralizing these bacterial toxins will limit thehost pro-inflammatory response and thereby improve outcome. Substancesemployed include antisera, polyclonal antibodies and monoclonalantibodies.

Because pre-clinical animal studies limited the host pro-inflammatoryresponse using both strategies, clinical trials for sepsis treatmentwere attempted. However, the clinical trials did not prove successfulexcept for low dose steroid treatment. These findings are summarized inTable 2. TABLE 2 Medicine Effect 1. Anti-Inflammatory TherapyGlucocorticoids Hydrocortisone large dose (100 mg i.v. then 0.18/kg/hr)none or worse small dose (100 mg i.v./every 8 hours) effective MediatorSpecific Anti-Inflammatory Therapy Anti-TNF-α Monoclonal antibody toTNF-α none Soluble TNF-α receptor antagonist none or worseAnti-Interleukin-1 Interleukin-1 receptor antagonists none OtherMediator Specific Anti-Inflammatory Therapy Anti-PAF PAF ReceptorAntagonist none Anti-Bradykinin Bradykinin Antagonist noneAnti-Prostaglandin Cyclooxygenase Inhibitor none 2. Anti-EndotoxinTherapy Antiserum none Polyclonal Antibody none Monoclonal Antibody none

Furthermore, the mortality rate from sepsis is high (35-50%), in spiteof this steroid treatment. It has been an important clinical priority tofind an effective sepsis therapy.

The reasons for these findings might be a failure to monitor howanti-inflammatory agents act on each step of inflammation in thepre-clinical studies.

There is a major medical need for a treatment that can be used not onlyfor treatment of chronic brain inflammation, but also on an acute andsubacute basis, to treat, prevent or inhibit the development of braininflammation. Furthermore, there is a major medical need for a treatmentthat can be used not only for treatment of chronic sepsis, but also onan acute and subacute basis, to treat, prevent or inhibit thedevelopment of sepsis. These are very serious, important, and unmetmedical needs. The ideal compound or compounds would have minimal sideeffects.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF INVENTION

It is an object of the present invention to overcome deficiencies in theprior art, such as indicated above.

It is also an object of the present invention to provide a method fortreating or inhibiting the development of a disease, disorder orcondition which is associated with increased capillary permeability andwhite blood cell extravasations and selected from brain inflammation andsepsis, or the symptoms thereof, in a mammal in need thereof, where themethod involves administering to a mammal in need thereof atherapeutically effective amount of a leukotriene C4 and D4 receptorantagonist.

The present invention involves the use of drugs that act as “leukotrieneantagonists”, viz., leukotriene C4 and D4 receptor antagonists.Accordingly, the present invention provides a method for short-term andsemi-long-term and chronic yet safe administration of a drug that cantreat, inhibit and/or prevent the development of brain inflammation andsepsis, or ameliorate the symptoms thereof, by administration of atherapeutically effective amount of a leukotriene C4 and D4 receptorantagonist, with the proviso that the brain inflammation is not focalcerebral ischemia.

The present invention also provides a method for screening an inhibitorof increased capillary permeability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the mechanism of action for sepsistherapies.

FIG. 2 is a graph depicting the action of ONO-1078 (pranlukast) ondextran-induced edema.

FIG. 3 is a schematic of the position of the cannula when measuringchanges in the CSF of rats during the sepsis experiments.

FIG. 4A and 4B are graphs showing that the administration of arachidonicacid (3.25 μg/2 μl) causes inflammation in the central nervous system oftwo different individuals. FIG. 4A is rat #1 and FIG. 4B is rat #2.

FIG. 5 is a graph showing the inhibitory effect of pranlukast (450mg/kg, i.p.) on the inflammation caused by arachidonic acid (3.25 μg/2μm) in the rat central nervous system (CNS). The data was obtained fromfour rats.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating or inhibiting thedevelopment of a disease, disorder or condition, which is associatedwith increased capillary permeability and white blood cell extravasationsuch as brain inflammation and sepsis, or for ameliorating the symptomsthereof, in a mammal in need thereof. The method involves administeringto the mammal a therapeutically effective amount of a leukotriene C4 andD4 receptor antagonist, preferably pranlukast, or a pharmaceuticallyacceptable salt thereof, which does not cross or minimally crosses theblood brain barrier.

The term “leukotriene antagonist” is used herein in the conventionalmedical sense to refer to a drug that suppresses, blocks, or otherwisereduces or opposes the concentration, activity, or effects of one ormore subtypes of naturally occurring leukotrienes. A leukotrieneantagonist typically antagonizes the actions of leukotriene at thereceptor level.

The present inventor has studied the inflammation mechanism with the useof leukotriene C4 and D4 receptor antagonists, in particular,pranlukast. Cyclooxygenase inhibitors (such as aspirin) are classifiedas Nonsteroidal Anti-inflammatory Drugs (NSAIDs) and are well known as aperipheral analgesics. However, it is difficult to show anti-edemaeffects of such cyclooxygenase inhibitors by measuring displacementwater in rat paw edema method. It is known that pro-inflammatorycytokines (TNF-α interleukin-1β) and interleukin-6), inflammatorymediators and toxins produced by bacterias and/or viruses causeinflammation in sepsis. However, their corresponding antagonists ofcytokines, anti-PAF, anti-bradykinin, NSAD and anti-endotoxin failed toinhibit the inflammation (Table 2).

Generally, when an inflammatory response develops, various cytokines andother inflammatory mediators act upon the local blood vessels, andincrease the expression of endothelial CAM. It is believed that thesepro-inflammatory cytokines and inflammatory mediators activatep-selectin and e-selectin on the endothelial cells of the capillary andthen start the process of white blood cell extravasation thatsequentially follows the steps of (1)Rolling, (2)Triggering,(3)Arrest/Adhesion, and (4)Transendothelial Migration.

TNF-α interleukin-1 and interleukin-6 produced by activated macrophagesat the site of the nociceptive stimuli play a particularly importantrole in mediating acute-phase inflammation as well as sepsis. In fact,proinflammatory cytokines administered to the outside of the blood brainbarrier through the chronically in-dwelling intracerebroventricularcatheter attached to an Ommaya reservoir increase white blood cell count(characterized by neutrophilic response and lymphocyte respectively) inthe cerebrospinal fluid in dogs. The inflammation induced byinterleukin-6, was longer lasting (starting from 2 hours, peak time are10-24 hours lasting 24-72 hours) than that induced by interleukin-1β andTNF-α. In the present studies presented in the Examples hereinbelow, theinflammation caused by interleukin-6 was used as a sepsis model,measuring the amount of infiltrating white blood cells in thecerebrospinal fluid.

Of particular interest, the present inventor discovered that pranlukast(300 mg/kg, 500 mg/kg, 1000 mg/kg), which was orally administered 30minutes before interleukin-6 was administered, completely inhibitedinterleukin-6 induced leukocyte in CSF for three days in the dog. Inspite of the presence of interleukin 6 in the cerebrospinal fluid, theadministration (p.o.) of pranlukast 30 min before interleukin 6completely inhibited brain inflammation for 3 days. Furthermore,pranlukast inhibited brain inflammation induced by the administration ofarachidonic acid in the subarachnoid space that increased capillarypermeability and then, after 0.5-1 hour, also increased theextravasation of white blood cells to the CSF and the brain parenchymain rats. These finding suggest that leukotriene C₄ and D₄ at the insideof the capillary plays an important role in the genesis of inflammation.

The process of inflammation is known as follows:

1) Macrophages activated by nociceptive stimulus secret TNF-αinterleukin-1β and interleukin-6 in tissues.

2) Vascular endothelial cells increase the expression of e-selectin byTNF-α and interleukin-1β, and p-selectin by histamine and thrombin inthe capillaries.

3) Circulating white blood cells express mucins such as PSGL-1 or thetetrasaccharides sialyl Lewis and sialyl Lewis^(a) and sialyl Lewis^(x),which bind to e- and p-selectin. This binding mediates the attachment ortethering of white blood cells to the vascular endothelium, allowing thecells to roll in the direction of the blood flow (Rolling). Althoughp-selection and sialyl Lewis-dependent alterations are induced byleukotriene C₄/D₄ in the mid-jejunum of rats, the leukotriene receptorantagonist was not effective (Samina Kanwar et al., (1995). Since then,potent selectin inhibitors were developed; however, they have not yetsucceeded (Alper J., 2001).

4) As white blood cells roll, chemokines (interleukin 8, MIP-1α/β, andMCP-1) and PAF, the complement split (C5a, C3a and C5b67) and variousN-formyl peptide are produced. Binding of these chemoattractants toreceptors on the white blood cell membrane triggers an activating signalmediated by G-proteins associated with the receptor. This signal inducesa conformational change in the integrin molecules in the white bloodcell membrane, increasing their affinity for immunoglobulin-superfamilyadhesion molecules on the endothelium (Triggering).

(5)Subsequent interaction between integrin andimmunoglobulin-superfamily CAMs stabilizes adhesion of the white bloodcell to endothelial cell, enabling the cell to adhere firmly to theendothelial cell(Arrest/adhesion).

The white blood cell then migrates through the vessel wall into thetissue. The steps in transendothelial migration and how it is directedare still largely unknown. They may be mediated by molecules bindingbetween the surface of white blood cell and CD31 on the capillaryendothelial cell, or by the binding LFA-1 on the white blood cells andJAM on the capillary endothelial cell (Transendothelial Migration).

Thus, white blood extravasation is a sequential reaction and that iscaused by interaction among capillary endothelial cells, molecules onthe capillary endothelial cells, chemokines and adhesion molecules(selectin family, integrin family, and immunoglobulin-superfamily).However, it has not been previously known that leukotriene C₄ and D₄play any role in the white blood cell extravasation in the centralcapillary.

The site of action of the leukotriene C₄ and D₄ receptor antagonistpranlukast is in the inside of the capillary lumen (involving thecapillary endothelial cells) that is covered with the blood brainbarrier in the central capillary.

Because pranlukast either does not cross or only minimally crosses theblood brain barrier, the anti-inflammatory effect of pranlukast is dueto the inhibition of increased capillary permeability and the inhibitionof white blood cells extravasation in the capillary lumen that involveadhesion of white blood cells to capillary endothelial cells, and thetransendotherial migration of white blood cells. Thus, pranlukastinhibits brain inflammation without crossing the blood brain barrier. Inthe case of a brain inflammation that has increased the permeability ofthe blood brain barrier, pranlukast in the plasma can pass though theblood brain barrier which has increased the permeability and bedelivered to the inflamed region.

In the general capillaries, the site of action of pranlukast isbasically the same as that in central capillaries i.e., pranlukast hasan anti-inflammatory effect through inhibition of white blood cellextravasation in the capillary lumen, involving adhesion of white bloodcells to capillary endothelial cells, and the transendothelial migrationof white blood cells. However, it may be distributed more widely in theperipheral tissues because the structure of general capillaries has moreopenings such as intracellular cleft, pinocytosis, and fenestra,compared to the structure of central capillaries.

Taken together, these findings and the previously disclosed data inTable 2 show that leukotriene C₄ and D₄ plays a more important role thanproinflammatory cytokines, inflammatory mediators in the white bloodcell extravasations in the capillary lumen involving adhesion of whiteblood cells to capillary endothelial cells, and the transendotheialmigration of white blood cells in both brain inflammation and systemicinflammation. The leukotriene C₄ and D₄ receptor antagonist, pranlukast,competitively antagonizes the leukotriene C₄ and D₄ receptor andeffectively inhibits brain inflammation (central inflammation) andsepsis (systemic inflammation).

The present inventor also discovered that dextran-induced rat paw edemawas inhibited by pranlukast in a dose-dependent fashion. At a dosage of450 mg/kg, administered intraperitoneally, pranlukast completelyinhibited dextran-induced paw edema. This suggests that leukotriene C₄and D₄ receptor antagonist acts at the endothelial cells in thecapillaries and inhibits the increased permeability of the capillarieswhich is induced by dextran. In spite of the many openings such asclefts, fenestrae and pinocytotic vesicles in endothelial cells in thegeneral capillary, pranlukast was found to inhibit peripherally thepermeability. Since the endothelial cells in the brain capillaries havefewer openings because of the presence of the tight junctions,pranlukast may be more effective in inhibiting the permeability of braincapillaries than that of general capillaries. Therefore, it was expectedthat such a mechanism might also come into play at the central nervoussystem level.

To investigate the role of leukotriene C₄ and D₄ receptor antagonist inthe treatment of brain inflammation, a sensitive and quantitative methodto measure inflammation for the central nervous system was developed bythe present inventor. According to this method, important changes in theinflammatory process induced by arachidonic acid can be monitored asfollows: 1)the changes in permeability of blood-brain barrier (bymeasuring the amount (volume(μl)) of cerebrospinal fluid resulting fromplasma component leaking through the blood brain barrier into thecentral nervous system)and 2) the infiltration of white blood cells intothe cerebrospinal fluid (by counting the number of white blood cellsusing the hemacytometer) over time from the same experimental animal. Inparticular, the effect of the leukotriene C₄ and D₄ receptor antagonist,pranlukast, on brain inflammation was studied using this method. Theadministration of arachidonic acid as a nociceptive stimulus tocerebrospinal fluid showed the important changes in the inflammatoryprocess of both the permeability of the blood brain barrier and theinfiltration of white blood cells through the blood-brain barrier.Pranlukast inhibited these changes induced by arachidonic acid. It isknown that leukotriene C₄ and D₄ receptor antagonists, such aspranlukast and zafirlukast as well as montelukast, either do not crossor minimally cross the blood-brain barrier. The present inventor has nowconfirmed that pranlukast acts directly or indirectly on endothelialcells in the blood brain barrier and inhibits capillary permeability toprevent leakage of plasma into the cerebrospinal fluid, and/or acts onthe capillary lumen and inhibits white blood cell extravasationsinvolving Rolling, Triggering, Arrest/adhesion and TransendothelialMigration. In the case of brain inflammation which results in increasedpermeability of the blood brain barrier, pranlukast in the plasma candistribute to the outside of brain capillary and can also act on theinflamed region in the central nervous system by crossing the bloodbrain barrier to inhibit brain inflammation. These findings aredifferent from the widely held concept that the inhibitor for braininflammation must cross or modulate the blood brain barrier for deliveryto the inflamed region.

The theory behind why leukotriene C4 and D4 receptor antagonists areinhibitors of increased capillary permeability and would therefore beuseful for treating sepsis is presented in FIG. 1. As a leukotriene C4and D4 receptor antagonist, pranlukast may be thought of as a new typeof an anti-inflammatory drug (anti-inflammatory therapies (2) shown inFIG. 1) that acts on endothelial cells at the post-capillary venulaand/or inside the post-capillary venula and provides a new therapy forsepsis that inhibits both central and peripheral inflammation.

The leukotriene C4 and D4 receptor antagonist pranlukast has been foundto be safe (LD₅₀>2000 mg/kg (p.o.) and (s.c.) in both rats and mice).After single administration of pranlukast (30 mg/kg, 100 mg/kg, 300mg/kg, 1000 mg/kg; p.o.), and repeated administration of pranlukast (30mg/kg/day, 100 mg/kg/day, 300 mg/kg/day, 1000 mg/kg/day; p.o.) for threemonths and six months, rats showed normal behavior, changes in bodyweight, and food intake compared with those of the control group. Theresults of urine examination and histopathological examinations are alsonormal by the single administration and the repeated administration ofpranlukast. The maximum blood concentration of pranlukast (administeredat 20 mg/kg) was attained within one hour after the administration(p.o.) and maintained for at least 5 hours. However, no pranlukast wasobserved 24 hours after administration of such low doses of pranlukast(data from Ono Pharmaceutical Co., Japan).

Zhang et al. (2002) reported that brain damage was induced byreperfusion 30 minutes after the occlusion of the middle cerebral artery(MCA) and was evaluated 24 hours after the reperfusion. Pranlukast(0.003 mg/kg-1.0 mg/kg) administered intraperitonealy 30 minutes beforeMCA and 2 hours after reperfusion inhibited the death of brain cells.However, Zhang et al. did not study blood concentrations. Indeed, noblood concentration of pranlukast would be found at 24 hours after thereperfusion at the dose levels (0.003 mg/kg-1.0 mg/kg) used by Zhang etal.

Generally, the severest brain damage develops 48-72 hours after theprimary injury. This secondary injury is a result of the primary injuryand is more serious than the primary injury. In most cases, the primaryinjury cannot be avoided and the secondary injury also cannot beprevented because there is no medication for brain inflammation. Largedoses of pranlukast however stay longer in the blood over 24 hours andcontinuously inhibit the increased capillary permeability and whiteblood cell extravasations. Therefore, in order to avoid secondary injurya large dose of pranlukast needs to be administered. If the primaryinjury can be completely inhibited, then the secondary injury does notdevelop. Small doses (0.003-1.0 mg/kg) of pranlukast cannot inhibit thedevelopment of the secondary injury. Zhang et al (2002) reported that0.1 mg/kg of pranlukast was the most effective dose in their study. Thisdose is equivalent to 6-8 mg in humans (body weight of humans is onaverage 60-80 kg) and is too low to be effective. It is expected thatpranlukast at a dose of less than 100 mg/day does not provide asufficiently effective anti-inflammatory effect.

Severe brain damage can be attenuated with large doses of pranlukast.Brain inflammation caused by a large dose of arachidonic acid (6.25μg/2.0 μl) was not inhibited by pranlukast (450 mg/kg; p.o.) but wasattenuated (white blood cell count returned to normal (=zero) 36 hoursafter arachidonic acid. Thus, brain inflammation caused by a large doseof arachidonic acid increased capillary permeability and increased whiteblood cell extravasation for 5 days without pranlukast. Administrationof pranlukast (450 mg/kg) attenuated the brain inflammation and theduration of the brain inflammation).

As discovered by the present inventor, the inhibitors of braininflammation, which do not cross or minimally cross the blood brainbarrier, and the sites of action are:

-   -   (A) at the inside of the capillary, the pharmacological effect        is as an anti-inflammatory agent (inhibition of the increased        capillary permeability as well as the process of white blood        cell extravasation from capillary to the tissue).    -   (B) at the outside of the capillary, in case of inflammation        that has already increased capillary permeability, pranlukast is        distributed with the plasma to the inflamed region because of        the increased permeability of the blood brain barrier, and        subsequently inhibits the brain inflammation.

The leukotriene C₄ and D₄ receptor antagonist pranlukast in thecapillary inhibits inflammation even though there are pro-inflammatorycytokines and/or inflammatory mediators at the outside of the capillary.

The anti-inflammatory effect of leukotriene C₄ and D₄ receptorantagonist pranlukast that inhibits the increased capillary peameabilityand white blood cell extravasation from blood brain barrier into thecentral nervous system is superior to those of antagonists ofpro-cytokines (TNF-α, interleukin-1β and interleukin-6 and otherinflammatory mediators such as prostaglandin, PAF, and thromboxane).

As mentioned above, the anti-inflammatory effect of pranlukast inhibitsthe expression of p-selectin and/or e-selectin, leukocyte-specific celladhesion molecules (CAMs) on endothelial cells or antagonizesleukotriene C₄ and D₄ that play an important role during white bloodcell extravasasions in the capillary lumen.

According to the method of the present invention, a therapeuticallyeffective amount of a leukotriene C4 and D4 receptor antagonist or apharmaceutically acceptable salt thereof, which antagonist does notcross or minimally crosses the blood brain barrier, is administered to amammal, preferably a human, in need thereof to treat or inhibit thedevelopment of a disease, disorder or condition associated withincreased capillary permeability and white blood cell extravasation inbrain inflammation and sepsis, or to ameliorate the symptoms thereof.

When the disease, disorder or condition to be treated or inhibited, orthe symptoms thereof to be ameliorated, is brain inflammation, thepresent invention is not intended to include brain inflammation causedby focal cerebral ischemia when the leukotriene C4 and D4 receptorantagonist is pranlukast and is administered preferably in a dose thatis about 400 mg/day to 800 mg/day in a low dose less than 100 mg/day.When pranlukast is preferably administered in a dose of about 400 mg/dayto 800 mg/day then the brain inflammation need not exclude focalcerebral ischemia. It is intended that the disease, disorder orcondition includes all other brain inflammations such as brain edema,stroke, hemorrhage (cerebral hemorrhage, subarachnoid hemorrhage, andsubdural hematoma), Willis Circle occlusion syndrome, brain trauma(injury, trauma resulting from surgery and invasive brain operations),brain infection and encephalitis. In the case of encephalitis, theencephalitis may be primary encephalitis (by Japanese encephalitisvirus, herpes simplex encephalitis, etc.), secondary encephalitis (byinfluenza virus, measles virus, chickenpox virus, rubella virus, etc.),and other subacute and chronic encephalitis. Non-limiting examples ofencephalitis include acute disseminated encephalomyelitisitis,encephalomyelitisitis after vaccination, meningitis after vaccination,pululent meningitis, encephalomyelitis after infection, meningitis afterinfection, acute cerebellar ataxia, compression myelitis, acuteascending myelitis, acute myelitis, sclerosing myeitis, meningiticmyelitis, meningoenchephalitis, myelitis, myelomeningitis,ventriculitis, meningitis, chronic meningitis. The brain inflammation isnot particularly limited as long as it is due to leakage of white bloodcells or the like from brain capillaries into surrounding tissues.

According to the present invention, sepsis is intended to include notonly infectious diseases caused by bacteria, but also those caused byviruses. Food poisoning caused by various bacteria such as Salmonellaenteritidis, Clostridium botulinum, etc., and severe acute respiratorysyndrome (SARS) caused by Coronavirus are preferred examples. AsCoronavirus mutates rapidly, developing an effective vaccine isdifficult. Therefore, according to the present invention, a leukotrieneC4 and D4 receptor antagonist can be administered to a patient when thepatient comes down with SARS, thereby avoiding severe symptoms.Furthermore, brain inflammation due to an infection such as influenzaencephalitis, encephalitis due to West Nile virus, cerebral meningitisincluding arachnoiditis are also included as sepsis.

It will be appreciated by those of skill in the art that theadministration of the leukotriene C4 and D4 receptor antagonist in themethod of the present invention can be performed pre-operatively beforebrain surgery or an invasive brain operation to prevent or inhibit braininflammation or after the surgery or operation.

Furthermore, as the present invention inhibits not only increasedcapillary permeability but also increased white blood cellextravasation, the administration of the leukotriene C4 and D4 receptorantagonist can be repeated until the white blood cell count in thetreated subject reaches a normal level in the cerebrospinal fluid.

The leukotriene C4 and D4 receptor antagonist used in the method of thepresent invention includes pranlukast (ONO-1078; Ono Pharmaceuticals,Osaka, Japan), zafirlukast and montelukast or a pharmaceuticallyacceptable salt and/or hydrate thereof. Pharmaceutically acceptablesalts include alkaline metals such as lithium, sodium, potassium, etc.,alkaline earth metals such as magnesium, calcium, etc., and aluminum.Non-limiting examples of other suitable leukotriene C4 and D4 receptorantagonists include the compounds,8-[2-(E)-[4-(4-fluorophenyl)butyloxy]phenyl]vinyl]-4-oxo-2-[5-1H-tetrazolyl)-4H-1-benzopyransodium salt (MEN-91507; Menarini), CR-3465 (Rottapharm), KP-496 (KakenPharmaceutical),4-[6-acetyl-3-[3-[(4-acetyl-3-hydroxy-2-propylphenyl)thio]propoxy]-2-propylphenoxy]butanoicacid (MN-002; Kyorin Pharmaceutical; U.S. Pat. No. 4,985,585), and(R)-3-methoxy-4-[1-methyl-5-[N-(2-methyl-4,4,4-trifluorobutyl)carbamoyl]indol-3-ylmethyl]-N-(2-methylphenylsulfonyl)benzamide (MCC-847; Astra Zeneca; EP 531078).

According to the method of the present invention, the magnitude of aprophylactic or therapeutic dose of a leukotriene C4 and D4 receptorantagonist in the acute or semi-chronic management of a disease,disorder or condition will vary with the severity of the condition to betreated and the route of administration. The dose, and perhaps the dosefrequency, will also vary according to the age, body weight, andresponse of the individual patient. In general, suitable oral dailydosage ranges of leukotriene C4 and D4 receptor antagonists for mildtrauma or for administration prior to brain surgery or an invasive canbe readily determined by those of skill in the art. For example, see thePhysician's Desk Reference, 54th Edition, Medical Economics Company Inc.(2000) for suitable dosages presently used for known leukotrieneinhibitors for the treatment of asthma. For leukotriene C4 and D4receptor antagonists, the oral daily dose range can be appropriatelyselected according to the symptoms of the patient. Specifically forpranlukast, the acceptable dose is preferably 100 mg/day-2000 mg/day,more preferably 200 mg/day-1000 mg/day, and most preferably 400mg/day-800 mg/day.

In the case of pranlukast, there does not appear to be enough of ananti-inflammatory effect at a dose of less than 100 mg/day or 1mg/kg/day of doses level. For montelukast, the dose range is preferably5 mg/day-100 mg/day, more preferably 5 mg/day-50 mg/day, and mostpreferably 10 mg/day-20 mg/day. For zafirlukast, the dose range ispreferably 10 mg/day-300 mg/day, more preferably 20 mg/day-150 mg/day,and most preferably 40 mg/day-80 mg/day. For more severe trauma orsymptoms, higher doses may be administered.

It is further recommended that children, patients aged 65 years andolder, and those with impaired renal or hepatic function initiallyreceive low doses, and that they then be titrated up based on individualresponse(s) or the dose level in the blood. It may be necessary to usedosages outside these ranges in some cases, as will be apparent to thoseof skill in the art. Furthermore, it will be appreciated that theclinician or treating physician will know how and when to adjust,interrupt, or terminate therapy in conjunction with individual patientresponse.

The most common dosage form at present is an oral formulation for mostpresently available leukotriene antagonists because of the need todissolve in stomach acid. Dosage forms may include tablets, troches,capsules, gel caps, lozenges, and the like. Due to their ease ofadministration, tablets and capsules represent the most advantageousoral dosage unit form, where solid pharmaceutical carriers are employed.In cases where the patient is unconscious, administration may preferablybe by cannula to the stomach. If desired, tablets may be coated bystandard aqueous or nonaqueous techniques. At least one presentlyavailable leukotriene C4 and D4 receptor antagonist, i.e., Singular(montelukast), can be administered intravenously. Administration can becarried out once a day. Administration may be terminated when the whiteblood cell count in cerebrospinal fluid reaches a normal value.

A further aspect of the present invention is directed to a method forscreening an inhibitor of increased capillary permeability for a mammalthat does not cross or minimally crosses the blood brain barrier andthat acts on vascular endothelial cells of capillaries and can inhibitincreased capillary permeability and/or acts on the capillary lumen toantagonize white blood cell extravasations and prevent leakage of plasmacomponents and blood cells from capillaries into tissues. The methodincludes the steps of: (1) administering a potential candidate inhibitorcompound to a non-human mammal such as a rat, a mouse, a gerbil, a dog,a cat, a rabbit or monkey, before or after an inflammation-inducingagent is introduced into the subarachnoid space via a cannula insertedinto the subarachnoid space through the dura mater of the brain of thenon-human mammal; measuring the amount of cerebrospinal fluid collectedthrough the cannula; and then determining from the measured amount ofcollected cerebrospinal fluid if the potential candidate inhibitorcompound is an inhibitor of increased capillary permeability.

Examples of the inflammation-inducing agent include arachidonic acid,prostaglandin, thromboxane, histamine, yeast, LPS, dextran, bradykinin,carrageenan, leukotriene, TNF-α, interleukin-1β or interleukin-6. Amongthem, a low molecular weight compound such as arachidonic acid ispreferred. In an animal model for sepsis, TNF-α, interleukin-1β orinterleukin-6 is preferred for the inflammation-inducing agent. Further,a variety of substances are exemplified as the test substance mentionedabove, however, leukotriene C4 and D4 receptor antagonist such aspranlukast is preferable. The method for administering a potentialcandidate inhibitor compound to a non-human animal is not particularlylimited and can be selected appropriately from known administrationmethods such as oral administration, subcutaneous administration,intra-peritoneal administration, intravenous administration,intramuscular administration, nasal (nasal drop) administration,inhalation, sublingual administration, suppository administration, etc.,in view of, for example, chemical properties (such as lipid-solubility,etc.) of the potential candidate inhibitor compound. The assessment ofwhether a potential candidate inhibitor compound can act on endothelialcells covering the inside of the capillary lumen to inhibit theincreased capillary permeability and the infiltration of white bloodcells from the capillary to the tissue can be conducted by measuring anamount of collected cerebrospinal fluid (extravasations of white bloodcells that involves the adhesion of white blood cells to the capillaryendothelial cells and the infiltration of white blood cells to theoutside of the capillary). However, it is preferred that the white bloodcell count in cerebrospinal fluid be measured together with the amountof cerebrospinal fluid. In addition, it is preferred that thecerebrospinal fluid is collected over time, e.g., every 30 minutes orone hour, and to assess the values relative to the controls (noadministration of potential candidate inhibitor compound). Where theadministration of the potential candidate inhibitor compound inhibitsthe exudation of cerebrospinal fluid and inhibits the presence of whiteblood cells in cerebrospinal fluid such as observed in the controls,then the potential candidate inhibitor compound can be regarded as aninhibitor of increased capillary permeability and is useful as apreventive agent for inhibiting increased capillary permeability whenthe inhibitor compound is administered prior to the introduction of aninflammation-inducing agent, and as an ameliorating agent when theinhibitor compound is administered after the introduction of aninflammation-inducing agent.

Only the potential candidate inhibitor compound is administeredperipherally without any inflammation inducing agent to theinteracerebroventricule in the screening method mentioned above. Usingthis system where the in-dwelling interacerebro/ventricular catheterattached to Ommaya reservoir, whether the potential candidate inhibitorcompound crosses the blood brain barrier or not can be easily determinedby measuring the amount of the potential candidate inhibitor compound inthe cerebrospinal fluid over time with a known method such as ELISA orthe like. The determination method is very useful for screening medicalsubstances targeting treatment of the brain. In that case, a dog ispreferable to a rat from the standpoint that it is possible to acquire acertain amount of cerebrospinal fluid.

The mechanism of the action of pranlukast, as demonstrated by the datapresented below, stabilizes endothelial cells at the post-capillaryvenula in both the periphery and centrally and inhibits the permeabilityat the first phase in the process of inflammation. Inflammatoryresponses occur in three distinct phases, each apparently mediated bydifferent mechanisms:

-   1. An acute transient first phase—vasodilation and increased    capillary permeability.-   2. A delayed subacute second phase—infiltration of leukocytes and    phagocytic cells; and-   3. A chronic proliferative third phase—necrosis of cells followed by    substitution with glia cells in the central nervous system, and    degeneration and fibrosis in peripheral areas.

A leukotriene C4 and D4 receptor antagonist, preferably pranlukast, canbe used according to the present invention to treat or inhibit thedevelopment of brain inflammation and sepsis, and/or to ameliorate thesymptoms thereof. This compound inhibits both peripheral and centralinflammation due to its activity of decreasing the permeability ofendothelial cells at the post capillary venula, thereby preventing orinhibiting leaking blood components at the first phase of inflammation.This anti-inflammatory mechanism of action of leukotriene C4 and D4receptor antagonist is unique and can be clearly differentiated fromconventional anti-inflammatory therapies and anti-endotoxin therapies(FIG. 1).

Anti-inflammatory Effect of Pranlukast in Peripheral Inflammation

Pranlukast inhibits dextran-induced paw edema in rats in a dosedependent manner. The administration of arachidonic acid to thesubarachnoid space causes inflammation. However, pranlukast (p.o.)administered before arachidonic acid administration to the subarachnoidspace inhibits inflammation. This may explain why pranlukast iseffective and is different from anti-inflammatory therapies andanti-endotoxin therapies.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration and are not intended to be limiting ofthe present invention.

EXAMPLE 1 Peripheral Studies

The present inventor discovered that dextran-induced rat paw edema wasinhibited by pranlukast in a dose-dependent fashion. At a dosage of 450mg/kg, administered orally, pranlukast completely inhibiteddextran-caused paw edema (FIG. 2).

This suggested that LT antagonists might act at the endothelial cells inthe peripheral capillary and inhibit the increased permeability of thecapillary induced by dextran. In spite of the many openings such asclefts, fenestrae and pinocytic vesicles among endothelial cells in thecapillary, pranlukast inhibited the permeability of the capillary. Sincethe endothelial cells in the brain capillary have less openings thanother capillaries because of the presence of tight junctions, pranlukastmay be more effective in inhibiting the permeability of the braincapillary than the permeability of a general capillary. Therefore, it isexpected that such a mechanism might also come into play at the CNSlevel.

EXAMPLE 2 Measurement of Changes in the Inflammatory Process

To investigate the role of leukotriene antagonists in the treatment ofinflammation, a sensitive and quantitative method to measureinflammation for the central nervous system was developed. Importantchanges in the inflammatory process can be monitored, as both thepermeability of BBB and the infiltration of white blood cells (WBC) tocerebrospinal fluid (CSF) caused by arachidonic acid can be measuredover time from the same experimental animal. In particular, the effectof the leukotriene C₄ and D₄ antagonist, pranlukast, was studied usingthis method. Changes in the inflammatory process of both thepermeability of BBB and the infiltration of white blood cells (WBC)caused by administering arachidonic acid as a nociceptive stimulus wereobserved.

Materials and Methods

Male rats, approximately 350-400 g, were anesthetized with sodiumpentobarbital at a dose of 50 mg/kg intraperitoneally (i.p.). Animalswere then placed in a stereotaxic unit. Fur from the top of the head toneck was shaved. The study area was disinfected with 70% ethanolfollowed by a 10% providone-iodine solution. Skin and muscle over thecervical cord were dissected until cervical vertebrae were exposed. Ahole was made using a 23G3/4 needle passing through the dura via thecisterna magna. One end of a 2-3 mm PE10 cannula (0.011 “I.D., 0.007”thick, 0.024″ O.D., 6.5 cm long) was inserted into the subarachnoidspace at the cervical cord so as to avoid damage to neuronal tissue(FIG. 3). The cannula was fixed with cyanoacrylic glue to the peripheraltissue. The other end of PE10 was used for administration of anociceptive stimulus, such as arachidonic acid (AA; 3.25 μg/2μl) andsubsequently for collecting cerebrospinal fluid (CSF) every thirtyminutes thereafter during the 11-hour experimental period. The cannulafilled with CSF immediately after the cannulation.

Air was infused to inject AA to the subarachnoid space. The muscles andskin were closed with surgical staples. The rat was then removed fromthe stereotaxic unit and placed on a heat pad and continuouslymonitored.

In the interaction study, the leukotriene C4 and D4 antagonist,pranlukast, was administered (450 mg/kg, i.p.) thirty minutes beforeadministration of arachidonic acid (AA). If the effects of theanesthesia appeared to be wearing off, an additional 0.05-0.1 mlpentobarbital (50 mg/ml, i.p.) dose was administered. If at any timeduring the experiment, the animal appeared to be in pain or distress,the study was immediately stopped and the animal was euthanized.

After the experiment, the animal was euthanized by exposure to highconcentration of halothane gas in an enclosed jar in a chemical fumehood.

Results

The volume of CSF increased immediately after the administration ofarachidonic acid (3.25 μg/2μl) to the subarachnoid space. CSF volumepeaked within 3.5 hours (FIGS. 4A and FIG. 4B). It gradually decreased4-5 hours after the AA administration, but it was seen to remain at acontinuous level during the 11-hour observation period at this doselevel.

Infiltration of WBC was not seen within the first 30 minutes, which mayimplicate an acute transient phase of brain inflammation, but thenstarted to increase slowly (FIG. 4A and 4B). The changes of the totalWBC counts and the volume of CSF rise and fall in parallel thereafter.This suggests a delayed subacute phase that is most characterized by theinfiltration of leukocytes and phagocytic cells. Neither increased CSFnor WBC count was observed in the control animal. This was the normalstate, as the pressure in the central nervous system is very low (6-10mm Hg) and there are usually no WBCs in CSF.

Pre-treatment using the leukotriene C4 and D4 receptor antagonist,pranlukast (450 mg/kg i.p.) thirty minutes before AA administration(3.25μg/2 μl) completely inhibited the increase of CSF volume (n=4) (FIG. 5).This shows that pranlukast blocks an increase in the permeability ofendothelial cells in the brain capillaries, as well as the infiltrationof white blood cells.

EXAMPLE 3 Studies of the Effects of the Leukotriene Receptor Antagonist,Pranlukast

Evaluation of the Increases of the Permeability of Endothelial Cells inthe Brain Capillaries and WBC Infiltration to the CSF DuringInflammation

Using the above-described method, it is possible to measure the changesin permeability over time in the same animal. No other method has beenable to measure sepsis and inflammation over time using the sameanimals, and as a result, it has been necessary to sacrifice at leastfour animals (to attain statistical significance) at certain timeperiods. For example, if it is necessary to investigate the process ofsepsis every thirty minutes for ten hours (n=4 for every thirtyminutes), then 80 rats would need to be sacrificed. It is important tonote that this method provides information not only regarding thechanges in permeability of the brain capillaries, but also regardingchanges in the infiltration of WBC into the CNS.

To obtain data on WBC infiltration into the CNS using this method, onlyfour rats are required, avoiding the differences between individualanimals for different time periods. Only this method can measurequantitatively both the permeability of the brain capillary and WBCinfiltration into the CNS and can provide a more complete picture of theprocess of inflammation.

In the present study, the volume (μl) of cerebrospinal fluid (CSF) andwhite blood cell (WBC) counts in CSF was measured every thirty minutesfor 11 hours from the same rat. One side of the cannula (PE10) wasinserted into the subarachnoid space under pentobarbital anesthesia,avoiding neuronal damage (FIG. 3). If the brain tissue is damaged, thevolume of CSF and WBC increases immediately without the administrationof arachidonic acid. Since there are no WBC in the CSF in the normalrat, only rats that had no WBC in the CSF immediately after thecannulation were used. Another side of the cannula remained open and wasused for the administration of nociceptive stimuli (arachidonic acid,LPS, dextran, etc.) and subsequently for collecting the CSF every thirtyminutes during the experiment. The CSF volume was measured using amicropipette and WBC in the CSF were counted by means of ahemacytometer.

Arachidonic acid (3.2 μg/2 μl) increased the CSF volume immediatelyafter administration, where a maximum volume of 100-120 μl was reachedwithin 3.5 hours. Although the volume decreased thereafter, the CSFvolume of 60-80 μl was maintained for every thirty minutes during4.0-5.0 hours post-administration. WBC could not be detected for thefirst thirty minutes after the administration of arachidonic acid, butthen was observed to increase gradually. The increase or decrease of WBCwas followed by changes in CSF volume over time.

Pranlukast (450 mg/kg) was administered intraperitoneally thirty minutesbefore the arachidonic acid application into the subarachnoid space.Pranlukast completely inhibited the increase in CSF volume and in thepermeability of the brain capillary marked by WBC infiltration.

The method characterized the first and second inflammatory phases asmentioned above, an acute transient phase characterized by localvasodilation and increased capillary permeability (first phase) and adelayed, subacute phase, most prominently characterized by infiltrationof leukocytes and phagocytic cells (second phase).

The brain capillaries have a relative absence of pinocytotic vesicles, agreatly increased number of mitochondria, and the presence of tightjunctions in capillaries, unlike general capillaries which have clefts,fenestrae and prominent pinocytic vesicles. It is known that leukotrienereceptor antagonists pass the BBB at very minimal levels, if at all.This fact suggests that leukotriene receptor antagonists peripherallyinhibit the permeability of the endothelial cells in the braincapillary. The endothelial cells are closely juxtaposed to one anotherand form tight junctions. These endothelial cells on the “inside” braincapillaries are much less likely to leak compared to those on generalcapillaries, since general capillaries have cleft passages, fenestraeand prominent pinocytic vesicles.

The treatment of brain inflammation by the leukotriene C4 and D4receptor antagonist might be due to peripheral inhibition of thepermeability of the endothelial cells between the capillary lumen andthe BBB. Therefore, this ability to decrease brain capillarypermeability and to inhibit infiltration of WBC into the central nervoussystem may be more important therapeutically than the drug being able topass through the BBB or to open the BBB to allow drugs to access thebrain inflammation. Once open, the BBB permeability will increase andcontribute to the formation of brain edema as the pressure of the braincapillary is higher than the intercranial pressure. Furthermore,infiltration of blood components to the central nervous systempotentiates brain inflammation.

These methods provide new findings as follows:

-   (1) The inflammatory process (phase 1 and phase 2) can be measured    quantitatively over time by these methods; and-   (2) Pranlukast inhibits the second brain injury as well.

EXAMPLE 4 Effect of Pranlukast and Other Anti-inflammatory Drugs onModels of Traumatic Brain Injury in the Rat

Pre-treatment of pranlukast has been found to result in longer survivalin a rat ischemia model using a reversible 30 minute occlusion of bothcarotid arteries followed by reperfusion. One of 4-5 rats survived forone week when pranlukast (450 mg/kg i.p.) was administered 30 minutesbefore the occlusion.

Additionally, increased infiltration of WBC into the CSF caused byinterleukin-6 were inhibited by pretreatment of pranlukast (administeredat three different doses) in the intact dog.

It has now been discovered by the present inventor that the leukotrieneC4 and D4 antagonist, pranlukast (450 mg/kg. i.p.), completely inhibitsinflammation caused by arachidonic acid (3.25 μg/2 μl). Thisinflammation is the basis of increased permeability of the BBB andinfiltration of WBC into the CSF. Therefore, the leukotriene C4 and D4antagonist (pranlukast) is useful as a treatment of inflammation due todiseases, disorders and conditions such as stroke, brain trauma and theeffects of brain surgery.

In view of the above and what is shown in Table 2, there is aninflammatory mechanism at the inside of capillary venulas that iscontrolled more strongly by leukotriene C4 and D4 than by cytokines suchas interleukin 1β, TNFα and interleukin 6 at the outside of capillaryvenulas.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by references.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

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1. A method for screening an inhibitor of increased capillarypermeability, comprising: administering a potential candidate inhibitorcompound to a non-human mammal before or after an inflammation-inducingagent is introduced into the subarachnoid space via a cannula insertedtherein through the dura mater of the brain of the non-human mammal;measuring the amount of cerebrospinal fluid collected through thecannula; and determining from the measured amount of collectedcerebrospinal fluid if the potential candidate inhibitor compound is aninhibitor of increased capillary permeability.
 2. The method of claim 1,wherein the inflammation-inducing agent is selected from the groupconsisting of arachidonic acid, prostaglandin, thromboxane, histamine,LPS, dextran, bradykinin, carrageenan, leukotriene, TNFα, IL-1β, andIL-6.
 3. The method of claim 1, further comprising measuring the whiteblood cell count in the cerebrospinal fluid collected.